Universal multidetection system for microplates

ABSTRACT

An apparatus for optically analyzing a sample may include an imaging subsystem that images the sample, one or more analyzing subsystems that analyze the sample, a temperature control subsystem that controls a temperature of the atmosphere within the apparatus, a gas control subsystem that controls a composition of the atmosphere within the apparatus, and a control module that controls the various subsystems of the apparatus.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. application Ser. No.12/838,804 filed on Jul. 19, 2010, which is a divisional of U.S.application Ser. No. 11/802,831, filed May 25, 2007, which issued asU.S. Pat. No. 7,782,454, the disclosures of which are incorporated byreference in their entireties.

BACKGROUND

1. Field

Apparatuses and methods consistent with the present invention relate todetection systems, including the detection of fluorescence, absorbance,and chemiluminescence in samples placed in the wells of microplates.

2. Description of the Related Work

Multiple analytical instruments are used in laboratories to evaluatesamples under test that are placed into vessels of various shapes. Inthe past twenty years, a microplate format has become very popular, asit lends itself to testing many samples on a single matrix-stylereceptacle. The first detection systems for microplates were absorbancereaders. Later dedicated fluorometers were developed, followed byinstruments to measure chemiluminescence.

The range of assay chemistries and labeling technologies continues togrow. Currently employed detection methods include absorbance,multiplexed fluorescence and chemiluminescence, fluorescencepolarization (FP), time-resolved fluorescence (TRF), fluorescenceresonance energy transfer (FRET), quenching methods, and speciallydesigned labels with intensity and spectral responsiveness toenvironmental conditions. Along with this range of detection methods,users are conjugating an ever-growing array of organic and inorganiclabels for targets, ranging from small-molecule drug candidates toproteins and nucleic acids, and to subcellular structures and cells.

FIG. 1 illustrates the general structure of a related art multimodedetection system. As shown in FIG. 1, a typical system comprises a lightsource 10, an excitation spectral device 20, an optical module 30, ameasurement chamber 60 with samples 70, an emission spectral device 40,and a detector 50. There are two distinct types of related art multimodedetection systems: filter-based units and monochromator-based units.

Filter-based units, when offered with high quality filters incombination with dichroic mirrors, allow for measurements with very lowdetection limits. This is mainly due to a high signal level, which isachieved with the filters, in combination with a high signal-to-noiseratio, which is achieved by a high level of blocking of the unwantedradiation around the desired waveband. The transmittance of filters isroutinely over 50%, and this high level of transmittance can be achievedindependent of the wavelength. Therefore, a very broad spectral rangecan be covered equally well from the deep ultraviolet (UV) to theinfrared (IR), and the bandpass of the filter can be tailored to thespecific application.

However, the filter-based unit cannot obtain a spectral scan forexcitation or emission of the substance under investigation. A user mustknow upfront what substance he or she is working with and order anappropriate filter set. In addition, when working in the deep UV,filters tend to degrade when exposed to the UV radiation of the lightsource, due to solarization. Also, maintaining libraries of filters forthe full range of labels is prohibitively expensive, and appropriatecombinations are often not readily available for a given label,conjugation chemistry, target molecule, and assay condition. Further,the effects of these conditions are not always predictable based on thenominal spectra of the basic label.

Monochromator-based instruments offer a high level of flexibility interms of choosing the wavelengths and obtaining scans of excitation andemission spectra, thus allowing the user to work with unknownsubstances. This also permits optimization of the measurements forperturbations to the spectra of labels due to assay conditions,conjugation chemistries, and target molecules. Additionally, whenworking with real biological or biochemical samples, interfering signalsfrom other sample components may require optimization of excitation andemission wavelengths for the exact assay conditions.

The monochromators used in modern instruments are usually based ondiffraction gratings, and use a flat grating for dispersion and concavemirrors for focusing light, or concave gratings that combine dispersiveand focusing functions. Monochromators require order sorting filters toseparate high spectral orders, but in the range from 200 nm to about 380nm, no order sorting filters are needed. Therefore, there is no need forfilters that withstand UV radiation, and the solarization problem isavoided.

However, the response of the monochromator is not constant across thewavelength range. One can obtain a system with a good signal in the UV,the visible, or the IR; however, one cannot obtain a system with a goodsignal in all of the wavelength ranges in the same monochromator-basedunit. A usual compromise is to optimize the excitation monochromator inthe UV and to optimize the emission monochromator in the visible or IR,because the wavelength of the emission light shifts to the right withrespect to the wavelength of the excitation light.

In order to obtain low detection limits, the monochromator must havevery low stray light. A traditional way to achieve this in themonochromator-based system is to employ two stage monochromators. Theseare called double monochromators, and contain two single monochromatorsplaced in series. While this does result in very low stray light, thepenalty is a dramatic decrease in signal, especially in spectral regionswhere the response of the single stage monochromator is already low.There are several instruments in the field based on this method.

In terms of performance, the filter-based units achieve significantlylower detection limits in fluorescence intensity applications across thefull spectral range, and work significantly better with techniques suchas TRF, FP, and Homogeneous Time-Resolved Fluorescence (HTRF), all ofwhich require the strong signal provided by the filter-based units. Onthe other hand, the monochromator-based units provide the flexibility ofchoosing any wavelength and the ability to obtain a spectral scan, atthe expense of lower sensitivity.

U.S. Pat. No. 6,313,471 describes a method that combines bandpassfilters and monochromators in series in a detection system. In thismethod, the bandpass filter acts as a crude first stage monochromator.The instrument splits the full spectral range of interest into severalregions corresponding to the number of filters employed, and blocksradiation from adjacent regions by using additional filters. The singlestage monochromator that follows the bandpass filters then selects thewavelength of interest from this prefiltered range.

However, with a limited number of prefiltered regions, this method islimited in flexibility. If both the excitation and emission wavelengthsfall into one region, the method is not effective in achieving low straylight or high performance. True spectral scanning is not readilyaccomplished with this method. This limits its utility for spectralmeasurement and optimization under conditions of fine spectralperturbation.

A most recent advance in microplate instrumentation is a multi-detectionanalyzer. An example of this product is the Synergy line from BioTekInstruments. The included modalities are absorbance, fluorescence,luminescence, and fluid injection.

There is a desire to study cellular processes in microplates, and thusthe need to visually study the contents of the microwells. Accordingly,a synergistic effect would be obtained by combining in one instrumentthe ability to perform imaging of the wells of the microplates andreading modality, such as absorbance, luminescence, or high sensitivityfluorescence readings.

SUMMARY

Exemplary embodiments described herein overcome the above disadvantagesand other disadvantages not described above. Also, the exemplaryembodiments are not required to overcome the disadvantages describedabove, and an exemplary embodiment may not overcome any of the problemsdescribed above.

According to an aspect of an exemplary embodiment, there is provided adevice for analyzing one or more samples, the device including a supportfor a receptacle that holds a sample; an imaging subsystem that imagesthe sample; and an analyzing subsystem that analyzes the sample.

According to an aspect of an exemplary embodiment, there is provided asample analysis method including selecting at least one subsystem fromamong a plurality of subsystems of a sample analysis device thatexamines one or more samples, the plurality of subsystems comprising animaging subsystem that images the one or more samples and an analyzingsubsystem that analyzes the one or more samples; and controlling theselected at least one subsystem to perform an examination on the one ormore samples, the examination comprising an imaging operation of theimaging subsystem that images the one or more samples and an analyzingoperation of the analyzing subsystem that analyzes the one or moresamples.

According to an aspect of an exemplary embodiment, there is provided anon-transitory computer-readable medium having embodied thereon aprogram which when executed by a computer causes the computer to executea sample examination method, the method including selecting at least onesubsystem from among a plurality of subsystems of a sample analysisdevice that examines one or more samples, the plurality of subsystemscomprising an imaging subsystem that images the one or more samples andan analyzing subsystem that analyzes the one or more samples; andcontrolling the selected at least one subsystem to perform anexamination on the one or more samples, the examination comprising animaging operation of the imaging subsystem that images the one or moresamples and an analyzing operation of the analyzing subsystem thatanalyzes the one or more samples.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other aspects will become more apparent by describing indetail exemplary embodiments thereof with reference to the attacheddrawings, in which:

FIG. 1 illustrates a general structure of a multimode detection system;

FIG. 2 illustrates certain components of a Universal Multi-detectionSystem (UMS) according to an exemplary embodiment of the presentinvention;

FIG. 3 illustrates a light source according to an exemplary embodimentof the present invention;

FIG. 4 illustrates an excitation spectral device according to anexemplary embodiment of the present invention;

FIG. 5 shows optical connections between the light source, theexcitation spectral device, and the excitation-emission separationdevice according to an exemplary embodiment of the present invention;

FIG. 6 illustrates an excitation-emission separation device according toan exemplary embodiment of the present invention;

FIG. 7 illustrates a holder according to an exemplary embodiment of thepresent invention;

FIG. 8 shows a view of the excitation-emission separation device alongvertical axes toward the microplate according to an exemplary embodimentof the present invention;

FIG. 9 illustrates an emission spectral device according to an exemplaryembodiment of the present invention; and

FIG. 10 shows a fluid dispenser according to an exemplary embodiment ofthe present invention.

FIG. 11A illustrates a UMS according to an exemplary embodiment, inwhich an imaging subsystem is included in the UMS.

FIG. 11B illustrates a UMS according to an exemplary embodiment, inwhich an imaging subsystem is included in the UMS.

FIGS. 12 and 13 illustrate a UMS according to an exemplary embodiment,in which atmospheric control is implemented.

FIG. 14 illustrates an LED module of an imager, according to anexemplary embodiment.

FIG. 15 illustrates a turret having objectives of the imaging module,according to an exemplary embodiment.

FIG. 16 illustrates views of a gas control module, according to anexemplary embodiment.

FIG. 17 is a functional block diagram that illustrates control ofmodalities, according to an exemplary embodiment.

FIG. 18 is a flowchart of a control method of a UMS, according to anexemplary embodiment.

FIG. 19 illustrates a control apparatus for controlling a UMS, accordingto an exemplary embodiment.

FIG. 20 illustrates operations of an imaging subsystem of a UMS,according to an exemplary embodiment.

FIG. 21 illustrates mode selection of operations of a UMS, according toan exemplary embodiment.

FIG. 22 illustrates parameter settings of an imaging subsystem of a UMS,according to an exemplary embodiment.

FIG. 23 illustrates parameter settings of an analysis subsystem of aUMS, according to an exemplary embodiment.

FIG. 24 illustrates parameter settings of an analysis subsystem of aUMS, according to an exemplary embodiment.

FIG. 25 illustrates settings of multiple subsystems of a UMS, accordingto an exemplary embodiment.

FIG. 26 illustrates monitoring operations by an analysis subsystem andconditional imaging by an imaging subsystem of a UMS, according to anexemplary embodiment.

FIG. 27 illustrates results of operations of an analysis subsystem andan imaging subsystem of a UMS, according to an exemplary embodiment.

FIG. 28 illustrates a detailed result of operations of an analysissubsystem of a UMS, according to an exemplary embodiment.

FIG. 29 illustrates data reduction options for combined analysis andimaging subsystems of a UMS, according to an exemplary embodiment.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Cell-based and live-cell assays are becoming more and more popular inlife science research and drug discovery as the field of biology keepsdeveloping in depth and complexity. Cells are complex biologicalentities and multi-parametric, multiplexed assays are becoming morecommon (for example, assay measuring 3 cellular events in parallel). Theability to monitor live cells using multi-parametric assays is key todeveloping a better understanding of cell biology.

Assays may be conducted with microplate multimode readers, using filtersor monochromators, which collect as much signal as possible from themicroplate well using the full population of cells. These assays aretypically quantitative and the signal is an average produced by the fullpopulation of cells. Other assays use microplate imaging readers thatcontain microscope objectives and a camera to image a small portion ofthe well and thus a sub-population of cells. This provides the abilityto localize where the signal is originating in individual cells andprovide semi-quantitative information from the extent of the signalcompared to controls. Another way of differentiating these assays iswhether they involve end-point or kinetic responses.

One benefit of a hybrid instrument is to dramatically increase theability to get multi-parametric data from live-cell assays. Inparticular, this novel instrument design allows making population-baseddetection synchronous with single-cell results available from imaging.2D imaging information produces feedback about individual cell behaviorswithin the population. This allows the simultaneous study of populationsand single cells, which will identify sub-populations and bettercharacterize the biology and drug effects.

This important benefit is especially evident in the case of kineticassays. Cellular events occur over a certain period of time (minutes,hours or days depending on type of event) and kinetic monitoring allowsrecording changes over time. Only a hybrid system as described hereinwill allow monitoring in parallel population-based information with themicroplate reader optics and single cell-based information with theimaging optics, providing a very detailed view of what is happening inthe sample. Such an assay may be one in which cell viability and celldeath are measured. This assay is typically measured with standard platereaders and the instrument acquires a signal coming from the solution inwhich the cells grow. Such a signal cannot be satisfactorily measuredusing imaging optics. On the other hand, these indirect signals areassumed to correspond to cell death and cell viability events but onlydirect visual inspection can confirm what is happening at the cellularlevel. Monitoring both chemical signals in solution with the platereader optics and actual cell morphology changes with the imaging opticsenhances the quality of the data set and provide more information on theactual cellular events.

Another unique benefit of a hybrid microplate reader defined as both amicroplate multimode reader and microplate imaging reader is significantinstrumentation cost savings and workflow improvement. This is due tothe ability to automatically perform both end-point and kinetic assayson both sub- and full populations of cells in the same sample, duringthe same experiment. Normally, this would require at least two separateinstruments. An example of this would be where a GFP fusion protein withHistone H3 is created using the Bacmam transfection technology. Histonesare located exclusively in the cell nucleus, so imaging ofsub-populations of cells using the imaging part of the hybrid reader isa useful validation step to ensure that green fluorescence from GFP islocated exclusively in the nucleus. The determination of the extent ofhistone deacetylation at the lysine 9 residue of histone H3 is thenconducted as an end-point assay using a labeled antibody against histoneH3 (lysine 9) on the full population of cells in the well using theoptics of a microplate multimode reader in the hybrid instrument.

Yet another unique benefit is the ability to improve on two majordrawbacks of imaging: read speed and amount of data generated. Imagingtakes longer than acquiring one data point per sample, and eachindividual sample file can be 1 MB or more. An instrument in accordancewith aspects of the present application will allow quickly scanningdozens or hundreds of samples, identifying the samples of interest thenlimiting imaging to these identified samples. This process willsignificantly reduce the total acquisition time and as well as the finalsize of the data set.

The combination of an imaging subsystem and an analyzing subsystempermits benefits in many areas, including those already discussed andthose discussed below.

a. Cell Counting

The purpose of cell counting in microplate assays is to estimate theincrease or decrease of a cell population after treatment with amolecule of interest. Cytotoxicity assays are intended to measurepopulation decline while cell proliferation assays are designed todetect population growth. These assays are among the most commoncell-based microplate assays run in life-science laboratories. Inconventional microplate readers, cell counting is performed using assaysthat generate a signal proportional to the number of cells. One of themost common such assay is a luminescent ATP assay: at the time ofmeasurement, the cells are lysed (the cell membrane is destroyed so thatthe content of the cell is released), and ATP (energy-storage moleculefound in all living cells) concentration is measured using a reagentthat generates light in the presence of ATP. ATP concentration isproportional to the number of cells, and as a result the luminescentsignal is proportional to the number of cells prior to analysis. Sincethis is a destructive assay, it can only be run once on each sample.There are instances where this type of assay can produce unexpectedresults.

The imaging subsystem permits scientists to take a quick look under amicroscope before processing the microplate, to ensure that that cellpopulation looks as expected. Accordingly, a second data point may beobtained for data analysis. Thus, for each sample, the user would havetwo sets of data: an image giving qualitative and semi-quantitative(estimate) information about the cell population, and a quantitativesignal once the ATP assay has been run. The two sets of data would beexpected to match in most cases (confirmation test), but a disagreementbetween the image and the quantitative assay would be a critical pieceof information.

b. Transfection Efficiency and Gene Expression Assays

The combination of an imaging subsystem and an analyzing subsystemenables documenting the history of the cell population using onesoftware and one instrument during transfection, which is a practice inmodern laboratories to add genetic material to cells for the purpose ofstudying specific genes.

For example, once the transfection has been accomplished and cells havehad time to recover from the process, the efficiency of the transfectionstep is estimated using the image subsystem to determine how many cellsamong the total cell population have effectively incorporated the newgene. Once this has been established, and if the transfection efficiencyis high enough, researchers can then carry on with their experiments andwill often run assays on conventional microplate readers.

c. Sample Documentation

The combination of an imaging subsystem and an analyzing subsystemenables sample information (qualitative and quantitative) to beregrouped in one electronic file, which will give researchers better andeasier access to their sample data, allow researchers to easily seemulti-dimensional data related to one sample, and thereby help bringclarity to the data-heavy cell-based research environment.

For example, large numbers of assay may rely on fluorescently labeledbiological samples (cells, tissues, microscopic worms and fish, . . . )to study the mechanisms of life. The imaging subsystem enables measuringdata points on an entire population or organism by measuring the totalfluorescence coming from a specific sample, which provides aquantitative answer. Further, qualitative data may be obtained todetermine where fluorescence is located, whether the samples look asexpected (number of cells, shape, distribution . . . ), etc., whichpermits better documentation of the sample.

The foregoing and other benefits attributable to the present applicationare particularly important for “Systems Biology”, which is a concept atthe core of modern biomedical research and has grown significantly sincethe year 2000. Systems Biology focuses on complex interactions in livebiological systems, and relies heavily on a proper experimental model.The cell represents one of these models that is increasingly used tostudy and understand more complex Systems Biology questions that cannotbe answered by simple mix-and-read assays. For this reason, properinstrumentation and software tools are essential for biologists runningcell-based and live-cell assays.

When working on live cells, it is important to control gas levels, inparticular CO₂ concentration for the purpose of maintainingclose-to-physiological conditions. Cells being living organisms, theyare sensitive to, and can react to, changes in their environment, suchas changes in gas concentrations or changes in temperature conditions.Changes in the gas conditions and thus pH of growth media have even beenlinked to change in gene expression.

The ability of systems in accordance with aspects of the presentapplication to maintain stable, continuous conditions while switchingdetection technology from imaging, to filter-based, tomonochromator-based, allows monitoring multiple qualitative andquantitative cellular events while maintaining stable cell cultureconditions. A benefit for users is the ability to precisely control andmaintain optimal conditions on one instrument platform, instead ofhaving to manually move samples from device to device, which couldinduce unexpected experimental artifacts (e.g. cells reacting totemperature change, mechanical movements or gas concentration changeswhile being transported from one device to the other).

Thus gas control in accordance with aspects of the present applicationwill provide more reliable, physiologically relevant results in bothshort term and long term studies in which the same plate might be readmany times over a time course. In this case the plate could remain inthe instrument and not be subjected to multiple cycles of climate andgas changes.

FIG. 2 illustrates certain components of a Universal Multi-detectionSystem (UMS) 100 according to an exemplary embodiment of the presentinvention. As shown in FIG. 2, samples are dispensed into the array ofmicrowells 200 in the microplate 300. The microplate 300 is transportedby the carriage 310 into the measurement chamber 320, which may beincubated, and is positioned sequentially for measurements. The lightsource 400 generates excitation light. The excitation spectral device500 selects and transmits a narrow band of the excitation light. Thewaveband is typically between 5 and 40 nm wide. The excitation-emissionseparation device 600 directs the excitation light to the microwells200, and then separates the emission light generated in the samplewithin the microwells 200 from the excitation light. Theexcitation-emission separation device 600 transmits the emission lightto the emission spectral device 700, which transmits a narrow band ofthe emission light. The emission spectral device 700 should beconfigured to transmit as much emission light as possible, whileblocking as much excitation light as possible and maximizing thesignal-to-noise ratio. The detector 800 converts the emission light intoan electrical signal. Although the light source 400, the excitationspectral device 500, the excitation-emission separation device 600, theemission spectral device 700, and the detector 800 are shown as separatemodules, they can also be combined in a variety of ways.

As shown in FIG. 2, relay devices 910, 920, 930, and 940 provide opticalconnections between the light source 400, the excitation spectral device500, the excitation-emission separation device 600, the emissionspectral device 700, and the detector 800. The controller 1000 storesemission signals from samples in the microplate 300, analyzes theemission signals, computes parameters categorizing the opticalmeasurements, and sends commands to the light source 400, the excitationspectral device 500, the excitation-emission separation device 600, theemission spectral device 700, or the detector 800. The commands caninstruct the light source 400, the excitation spectral device 500, theexcitation-emission separation device 600, the emission spectral device700, or the detector 800 to change an internal parameter. For example,the commands can instruct the excitation spectral device 500 or theemission spectral device 700 to use one internal device instead ofanother internal device. Further, an optional dispenser 1100 deliversreagent to the microwells 200.

FIG. 3 illustrates the structure of the light source 400 according to anexemplary embodiment of the present invention. In a preferredembodiment, the light source 400 comprises only two light generatingdevices: a Xenon flash lamp 410 and a Tungsten lamp 420. In otherembodiments the light source 400 may comprise a Xenon continuous wavelamp, a light emitting diode (LED), a laser, or any otherlight-generating device.

Tungsten sources are very stable, and their radiation extends from bluein the visible spectrum to the far IR, and peaks around 1 μm. They aremost suitable for measurements in the visible and IR regions of thespectrum. In contrast, Xenon flash sources deliver most of theirradiation in the deep UV, UV, and short visible spectral ranges. Inaddition, Xenon flash sources provide a very fast burst of light,lasting for several microseconds with a fast decay, and are thereforesuitable for time resolved measurements in modern multi-detectionsystems.

The Xenon flash lamp 410 has a parabolic reflector 411 positioned suchthat the arc 412 of the lamp 410 is located near the focal point of thereflector 411, providing an essentially collimated beam from thereflector 411. The Tungsten lamp 420 has a parabolic reflector 421positioned such that the filament 422 of the lamp 420 is located nearthe focal point of the reflector 421, providing an essentiallycollimated beam from the reflector 421. FIG. 3 shows that a lens 423 maybe used to focus the beam from the reflector 421 onto the exit portal430 of the light source 400. As shown in FIG. 5, relay optics may beused to focus the beam onto the entrance of an optical fiber.Alternatively, the lens 423 may focus the beam from the reflector 421directly onto the entrance of an optical fiber within the excitationspectral device 500.

The movable off-axis parabolic reflector 440 has two working locations.In the first location, depicted by a solid line in FIG. 3, the reflector440 reflects and focuses light from the reflector 411. In the secondlocation, depicted by a dashed line in FIG. 3, the reflector 440 staysout of the way of light from the reflector 421. This arrangement allowslight from either lamp to be focused at the same location. Further, thefan 417 directs air across the fins 415 of a cooling extrusion for theXenon source 410 and onto the Tungsten source 420. This arrangementallows both sources to share a single cooling system.

The arrangement of two light sources in close proximity to each other,with their optical axes offset, and preferably at an angle ofapproximately 90 degrees to each other, allows for a very compactillumination system with a shared cooling system. The use of parabolicreflectors around the light sources, in combination with off-axisparabolic reflectors, results in very highly efficient coupling of lightfrom the arc and filament into the system. Here the final focusing pointof both light sources is the same. This system allows a more compactarrangement than a system which utilizes separate light sourcecompartments with separate exit light points for each compartment, thusrequiring a mechanical movement of the optical relay system to switchbetween sources.

FIG. 4 illustrates the structure of the excitation spectral device 500according to an exemplary embodiment of the present invention. The exitportal 430 of the light source 400 is in close proximity to the inputportal 510 of the excitation spectral device 500. The relay device 910is unfilled space inside the UMS 100.

In a preferred embodiment, the excitation spectral device 500 has twospectral selection devices, which differ by the physical technology bywhich they separate light with different wavelengths. The first deviceis a filter selection device 520, which has a variety ofuser-replaceable filters 521. The second device is a doublemonochromator 530.

As shown in FIG. 4, the light exiting the light source 400 via the exitport 430 is directed to the entry point 510 of the excitation spectraldevice 500. Light entering the excitation spectral device 500 is thendirected to the exit port 540 of the excitation spectral device 500along one of two paths.

The first path directs the light through one of the filters 521 in thefilter selection device 520, which transmits a narrow band of the light.The light then propagates through optical fiber 522 to the exit port540. The second path bypasses the filters 521 by directing the lightthrough hole 523 in the filter selection device 520. The light thencontinues via optical fiber 531, which accepts a circular image of thearc or filament spot from the light source 400 formed at the entry port510, and shapes the light spot into a slit shape to match it to theinput slit of the double monochromator 530. The monochromator 530selects a narrow band of the light, and then the optical fiber 532changes the shape of the light from the exit slit shape of themonochromator 530 into a circular shape that resembles the shape of amicrowell 200.

The light path selector 550 can move relative to the filter selectiondevice 520, providing the ability to guide light to the exit port 540that was spectrally selected by the filters 521 or the monochromator530. FIG. 5 shows the optical connections between the light source 400,the excitation spectral device 500, and the excitation-emissionseparation device 600 in greater detail.

FIG. 6 illustrates the structure of the excitation-emission separationdevice 600 according to an exemplary embodiment of the presentinvention. The general purpose of the excitation-emission separationdevice 600 is to irradiate the sample with excitation light and/orgather emission light from the sample. The excitation-emissionseparation device 600 can be positioned above the microwell 200 as shownin FIG. 6, or below the microwell 200. Also, the UniversalMulti-detection System 100 can include two excitation-emissionseparation devices 600, one of which is positioned above the microwell200, and the other of which is positioned below the microwell 200. Thisarrangement enables measurements of the same microwell 200 with afilter-based system and a monochromator-based system from both the topand the bottom.

In a preferred embodiment, several light paths may be used, based on themeasurement technique. For absorbance measurements, the excitation andemission light are preferably collinear. As shown in FIG. 6, theabsorbance measurements are conducted in block 640, in which themicrowell 200 is illuminated with excitation light from below at pointG. This excitation light may come from the monochromator 530 or thefilter selection device 520, as shown in FIG. 5. A detector 650 isplaced on the opposite side of the microwell 200 to capture emissionlight that passes through the sample.

For luminescence measurements, no excitation light is required, and onlyemission light is gathered from the sample by the excitation-emissionseparation device 600. In block 630, a single fiber optic bundle 735 isused to maximize the light gathering capability of the system and thusimprove the signal.

For fluorescence measurements, two optical paths are available toirradiate the sample with excitation light and to gather emission lightfrom the sample. These paths can be optimized to further enhance theoverall system performance.

Block 620 depicts a first optical path for fluorescence measurements,which can use a partially reflective mirror or a dichroic mirror so thatexcitation light and emission light are collinear when entering andexiting the sample, respectively. Light is delivered to Block 620 by theoptical fiber 522. The movable aperture 601 has several openings withdiameters preferably ranging from approximately 1.5 mm to 4 mm, and isplaced in front of the guide fiber 522. An image of the opening placedin front of the optical fiber 522 is formed in the microwell 200 bylenses 621 and 622. The size of the opening of the movable aperture 601is selected to fill the microwell 200 as completely as possible withlight, while preventing light from entering adjacent microwells andcausing cross-talk.

The light is reflected by a partially transmitting mirror 623 on amovable holder 627. More than one mirror can be placed onto the holder627. Some mirrors can be dichroic mirrors to improve the signal, as allexcitation light is reflected towards the microwell 200, and allemission light is transmitted towards exit fiber. The dichroic mirrorscan also improve the signal-to-noise ratio of the measurement system, asresidual excitation light that reaches the microwell 200 and isreflected by the meniscus lens is blocked from reaching the exit fiber.The emission light from the microwell 200 is gathered onto the fiberoptic bundle 731 by lenses 621, 622, and 670. A collective lens 670 infront of the fiber optic bundle 731 assures that emission light from thefull depth of the microwell 200 is collected, thus maximizing the systemsignal.

The high energy collection characteristics of the system assure lowdetection limits and allow for various levels of fluid to produceacceptable results without the need to refocus the optical system basedon the fluid volume. This is in contrast with, for example, the confocalstyle measurements described in U.S. Pat. No. 6,097,025, which uses aconfocal optical system that collects light only from the small portionof the microwell.

In a preferred embodiment, linear polarizers 624 and 625 are added tothe holder 627, and the same motion that positions appropriate mirrorsin the light path also can be used to select polarizers for fluorescencepolarization measurements. This eliminates the need for a separatemechanism to switch the polarizers, and thus improves the reliability ofthe system.

Block 610 depicts a second optical path for fluorescence measurements,which uses a tilted V arrangement of optics for direct well illuminationand light gathering. This allows the system to channel the full amountof light from the fiber optic 532 into the microwell 200. The numericalaperture of the optics 611 and 612 is matched to the fiber optic 532 forthis purpose. The cone of excitation light enters the microwell 200 andexcites the contents of the microwell 200 via the first leg of the V.The emission light is collected by the second leg of the V. Thenumerical aperture of lenses 614 and 613 matches the exit fiber optic732. The V is tilted with respect to the vertical plane to directexcitation light that is specularly reflected from the surface of themicrowell 200 away from the light collecting leg of the V. Therefore,this arrangement introduces a spatial separation of emission andexcitation light in addition to the spectral separation, andsignificantly improves the signal-to-noise ratio. This tilted Varrangement can also be used to conduct fluorescence polarizationmeasurements.

The entry ports A and B of the excitation-emission separation device 600accept fiber bundles from the excitation spectral device 500. Fibers canbe positioned to direct light that is spectrally separated by filters inthe excitation spectral device 500 into input B of Block 620. Fibers canalso be positioned to direct light spectrally separated bymonochromators in the excitation spectral device 500 into input A ofBlock 610. Alternatively the inputs can be reconfigured by switchingfibers 522 and 532. This switching may be accomplished manually. Theemission light is gathered by fibers 731 and 732 from ports C and D. Theplacement of fibers 731 and 732 in the exit ports C and D determines theorigin of the emission light in the fibers.

FIG. 7 illustrates the holder 627 with associated dichroic mirrors 623,628, and 629 and linear polarizers 624, 625, and 626 according to anexemplary embodiment of the present invention. The holder 627 is affixedto the slider 650, which slides along rail 651 due to the applied forcefrom the motor 652 through the belt 653. The holder 627 moves in adirection perpendicular to the plane defined by the optical axes of theexcitation and emission light. Although two different fibers 522 and 532could occupy the fiber position depicted in FIG. 7, for the sake ofclarity only fiber 522 is shown.

In the depicted design there are five possible positions for the holder627 relative to the fiber 522, which delivers the excitation light. Thefirst position, which is depicted in FIG. 7, represents a situationwhere the center of mirror 628 is aligned with the optical axis of thefiber 522. In this position fluorescence polarization based assayscannot be conducted. If the holder 627 is moved to the left for adistance equal to the distance between the centers of mirror 628 and629, the holder 627 will be in the second position. In the secondposition, the mirror 629 plays an active role, and fluorescencepolarization based assays cannot be conducted.

The three other positions of the holder 627 correspond to threedifferent situations. First, when the right third of the mirror 623 ispositioned in front of the fiber 522, fluorescence polarization basedassays cannot be conducted. Second, when the middle third of the mirror623 is positioned in front of the fiber 522, the linear polarizer 624 isin the optical path of the excitation light, and the linear polarizer626 is in the optical path of the emission light. In this case thepolarization vectors of the excitation and emission light are crossed.Third, when the left third of the mirror 623 is positioned in front ofthe fiber 522, the linear polarizer 624 is still in the optical path ofexcitation light, and another linear polarizer 625 is in the opticalpath of the emission light. In this case the polarization vectors of theexcitation and emission light are parallel. Thus the linear motion ofthe holder 627 not only selects which mirror is placed in the opticalpath, but also allows for fluorescence polarization measurements.

As shown in FIG. 7, the linear polarizers 625 and 626 have parallelsurface orientations and perpendicular polarization axis orientations.They have active areas of equal sizes, and each size is comparable tothe size of the cross-section of the emission light. The polarizationaxis of the linear polarizer 624 is parallel to the polarization axis ofthe linear polarizer 625, and perpendicular to the polarization axis ofthe linear polarizer 626. The area of the linear polarizer 624 is atleast twice the area of the linear polarizer 625. The area of the mirror623 is at least three time the area of the linear polarizer 625. Themirror 623 is partially reflective and partially transparent.

FIG. 8 shows a view from above the microplate 300, along vertical axestoward the microplate 300 of the block 610 of the excitation-emissionseparation device 600. Points A and B′ are input portals of theexcitation-emission separation device 600. Lenses 611, 612, 663, and 664focus excitation light onto the microwell 200 in the microplate 300.Lenses 613, 614, 673, and 674 collect emission light and focus it intopoints C and D′, which are exit portals of the excitation-emissionseparation device 600. Standard 384 well microplates have an upper edgewith a nearly square shape. The optical axes of lenses 611, 612, 663,664, 613, 614, 673, and 674 are oriented along the diagonals ofmicrowells 200. Using this arrangement a reading may be taken on thesame microwell 200 simultaneously via filter-based ormonochromator-based spectral systems. Because the excitation light frompoint A is reflected toward point B′ and vice versa, very littleexcitation light is reflected toward exit portals C and D′. Therefore,the emission light is spatially separated from the excitation light.

FIG. 9 illustrates the structure of the emission spectral device 700according to an exemplary embodiment of the present invention. In apreferred embodiment, the emission spectral device 700 has two spectralselection devices, which differ by the physical technology by which theyseparate light with different wavelengths. The first device is a filterselection device 710, which has a variety of filters 711. The seconddevice is a double monochromator 720. As shown in FIGS. 6 and 9, fibers731, 732, and 735 extend from their respective locations within theexcitation-emission separation device 600 into the emission spectraldevice 700. The selector switch 760 is used to direct light from fibersto the filter selection device 710. FIG. 9 shows that the fiber 731 fromfluorescent measurement block 620 and the fiber 735 from theluminescence measurement block 630 are connected to the selector switch760. FIG. 9 also shows that the fiber 732 from the fluorescentmeasurement block 610 is connected to the monochromator 720. However,the arrangement in FIG. 9 is merely exemplary, and a user can change theconnections by physically switching the fiber connections within theexcitation-emission separation device 600, or within the emissionspectral device 700.

In a preferred embodiment, the exit portals of the excitation-emissionseparation device 600 are in close proximity to the input portal 730 ofthe emission spectral device 700. Points E and F may represent the exitportals of the emission spectral device 700. The detector 800 maycomprise two photomultiplier tubes (PMTs) positioned at points E and F(not shown).

FIG. 10 shows a fluid dispenser 1100 according to an exemplaryembodiment of the present invention. The purpose of the fluid dispenser1100 is to inject fluid into microwells 200 to initiate the reactionunder investigation. Often the time from initiation to the timemeasurements have to take place is very short. Therefore, the injectionports 1101 and 1102 may be placed in close proximity to the opticalreading system. Further, two separate fluid lines 1111 and 1112 may beused. Each fluid line connects to the stepper motor driven syringe drivefor positive displacement fluid delivery. A three-way valve alternatelyconnects a syringe to supply bottles on a suction stroke or to aninjector line for dispensing.

In view of demand for investigating live cells, the multi-detectionsystems discussed below may further include the ability to imagecontents of the samples, along with obtaining quantitative data byfluorescence, absorbance, or luminescence, and the ability to provide inthe same multi-detection analyzer a controlled gas atmosphere forsamples preserving the long term viability of live cells.

FIG. 11A illustrates a UMS according to an exemplary embodiment, inwhich an imaging subsystem is included in the UMS.

The UMS illustrated in FIG. 11A includes a measurement chamber 320, anexcitation-emission separation device 600, an optional dispenser 1100,and an imaging subsystem module 1200. Samples are dispensed into thearray of microwells 200 in the microplate 300. The microplate 300 istransported by the carriage 310 into the measurement chamber 320, whichmay be incubated, and is positioned sequentially for measurements. Themicroplate 300 may be a matrix-styled receptacle for holding slides orwells of sample.

The imaging subsystem module 1200 has visual access to the microwells200 of the micro plate 300 located on the carriage 310 housed in themeasurement chamber 320. Further details of the imaging subsystem moduleare discussed later below with respect to FIGS. 12 and 13.

The imaging subsystem module 1200 includes an independent light source1201 such as, for example a full spectrum or single color light-emittingdiode (LED), that sends light via an epi-fluorescenceExcitation/Emission filter cube 1210 and mirror 1220 into a microscopeobjective 1230.

FIG. 15 illustrates a turret having objectives of the imaging module,according to an exemplary embodiment.

The microscope objective 1230 may be disposed on a turret 1240, whichrotates and/or changes position to change objectives and moves up anddown to focus the microscope objective 1230 onto the microwells 200.

By moving the turret with objectives vertically relative to microplate300, the focusing of the objectives onto the object of interest can beaccomplished. The same vertical motion allows for the entry of theobjective into incubation chamber 320 and removal of the objective outof the chamber when the imaging system is not in use.

As illustrated in FIG. 15, the rotating objective turret may be mountedon motorized vertical linear way that allows insertion of one of theobjectives at a time into incubation chamber and allow for focusing ofthe objective on the sample. A thermal barrier plug 1231 may beinstalled in addition to an objective 1230, and may be used to close theincubation chamber when imager is not being used for sampleobservations. Accordingly, maximized sample chamber temperatureuniformity may be obtained when imaging modality is in use or is notbeing used.

The image of the microwell 200 is imaged by tube lens 1250 and a camera1260.

FIG. 14 illustrates the design and positioning of the LED illuminationmodule 1201 and the filter cube 1210. The lower LED module 1201 mayinclude an LED and a focusing lens. The filter cube 1210 includes anexcitation filter 1211, a dichroic mirror or partial mirror 1212, and anemission filter 1213.

The coordination of elements of the imaging subsystem module 1200 andmotions of the microplate are controlled by a controller, which may beseparately embodied or combined with a controller 1000 of FIG. 2 thatalso controls operation of the other multi-detection reading modalities.The controller may be a processor (e.g., central processing unit,microprocessor) that executes instructions stored in a memory forperforming the imaging. The imaging of the microwell 200 contents canthus can be interlaced as part of an assay (steps in multimode plateprocessing discussed above). As a result, the samples that haveundergone processing may be imaged.

Implementation of the imaging subsystem module 1200 is not limited tothe configuration illustrated in FIG. 11A and FIG. 13 as discussedbelow. For example, the UMS 100 of FIG. 2 may be modified to furtherinclude the imaging subsystem module 1200. Similarly, the imagingsubsystem module 1200 may be incorporated into the various embodimentsof FIGS. 2 to 10 discussed throughout the application.

FIG. 11B illustrates a UMS according to an exemplary embodiment, inwhich an imaging subsystem is included in the UMS.

The UMS illustrated in FIG. 19 includes a measurement chamber 320, amonochromator based measuring system 12036 and filter based measuringsystem 12035, an optional dispenser 1100, and an imaging subsystemmodule 1200.

As illustrated in FIG. 11B, the measurement chamber 320, themonochromator based measuring system 12036, the filter based measuringsystem 12035, the optional dispenser 1100, and the imaging subsystemmodule 1200 are separate systems that can be independently controlledand manipulated.

Though the systems of FIG. 11B may be independently controlled, thesystems may be implemented in a common housing, as will be discussedbelow. Further, the systems may be controlled independently through acommon interface, as will also be discussed below.

Samples are dispensed into the array of microwells 200 in the microplate300. The microplate 300 is transported by the carriage 310 into themeasurement chamber 320, which may be incubated, and is positionedsequentially for measurements. The microplate 300 may be a matrix-styledreceptacle for holding slides or wells of sample.

Monochromator based measuring system 12036 could be used forfluorescence measurements, chemiluminescence measurements and absorbancemeasurements. The monochromator based measuring system 12036 may includea broad band light source 13001, double excitation monochromator 13002(stage 1) and 13003 (stage 2). The excitation light is delivered tosample via fiber optics 13005 and focusing lens 13020. The emissionlight is picked up by focusing lens 13020 and via fiber bundle 13005 isguided to the double emission monochromator 13010 (stage 1) and 1311(stage 2), and then to detector 13020. The emission path may also beused for chemiluminescence measurements. The excitation doublemonochromator may direct light via fiber 13030 to the absorbancemeasuring lenses 13040 and 13050 and onto detector 13060. The well 200is positioned under the light beam for a specific measurement.

The filter based measuring system 12035 may be used for fluorescencemeasurements, chemiluminescence measurements and absorbancemeasurements. The filter based measuring system 12035 may include lightsource 14001, excitation/emission cube 14010, focusing lens 14020, anddetector 14020. In case of absorbance measurements a lens 14040 focusesradiation that passed through the microwell 200 onto detector 14050.

The imaging subsystem module 1200 has visual access to the microwells200 of the micro plate 300 located on the carriage 310 housed in themeasurement chamber 320. Further details of the UMS including theimaging subsystem module are now discussed below with respect to FIGS.12 and 13.

FIGS. 12 and 13 illustrate a UMS according to an exemplary embodiment,in which atmospheric control is implemented.

In view of interest on the part of researches to work with live cellsand perform long kinetic studies, while obtaining both quantitative(i.e., fluorescence, absorbance, luminescence, etc.) information andqualitative information (i.e., imaging, etc.), it is important toprovide an environment for sample that is conducive to the cell's longterm viability. This may be accomplished by controlling gas atmospherearound the cell samples. As will be discussed below, the multi-detectionanalyzer of FIG. 12 allows combined quantitative modality, such asfilter or monochromator based optical measurements, along withquantitative imaging of samples, and the ability to conduct experimentsand obtain measurements in a controlled gas environment.

The UMS 12001 includes a single housing 12017 and a dual-purpose basestructure, which includes a base 12100 and a rear plate 12120. Thesingle housing 12017 creates one common atmosphere within the UMS. Thesingle housing 12017 is designed to be substantially, and preferablycompletely, gas tight. The plate 12100 supports elements of theequipment compartments and sample compartments, but the plate 12100 doesnot separate the atmosphere of the elements of the equipmentcompartments and the sample compartments. That is to say, the elementsof the equipment compartments and the sample compartments are subjectedto the common atmosphere created by the housing 12017.

The sample compartment may include a micro plate 12004, similar to themicroplate 300 discussed above, on carrier 12005 that is surrounded bytop incubator plate 12105 and bottom incubator plate 12110, and may notbe sealed by the top incubator plate 12105 and the bottom incubatorplate 12110 when inside the housing 12017. The microplate 12004 mayinclude microwells in which samples are contained.

The UMS 1201 includes a filter detection module 12035 and amonochromator detection module 12036. Both the filter detection module12035 and the monochromator detection module 12036 may contain motorsand light sources, which generate heat when operated. As testing on thesamples may require an ambient temperature, the heat generated by thefilter detection module 12035 and the monochromator detection module12036 should be effectively removed from the inside of the instrumenthousing 12017 to maintain temperature in the sample chamber close toambient, in particular when incubation of the sample is not required.Preferably, the generated heat may be removed without introduction ofair inside the instrument and discharge of the introduced air forcooling. Particularly, as the introduction of cooling air may carry dustparticulates, which may increase sample evaporation and introduce errorsin sample imaging and analysis.

The heat generated by the filter detection module 12035 and themonochromator detection module 12036 in the equipment compartment isconductively channeled via base 12100 to the rear plate 12120. Heattransferred through the base 12100 to the rear plate may be removed byforced convection by a fan 12130 that is external to the housing 12017.Thus, there is no need to introduce cooling air, the flow of which andthe potential contamination by which makes incubation of samplesdifficult, inside the housing 12017.

As a result, temperature within the housing 12017, and thus temperatureof the sample compartment may be controlled.

The base of the imaging module 1200 may be attached to the same baseplate that holds filter module 12035, monochromator module 12036, andmicroplate transmission module 310.

In addition to temperature control, composition of the atmosphere mayalso be controlled by a gas controller module, an overall view of whichis illustrated in FIG. 16.

Referring to FIGS. 12 and 13, gas introduced from gas module 12006 vialine 12038 and injector 12021 is dispersed via fan 12022. The lines12038 may also include a sampling gas line coming to gas module 12006,which regulates the gas mixture delivered to the system by injector12021. The gas module may be separately embodied or combined with acontroller 1000 that may also control operation of the othermulti-detection reading modalities. The introduced gas fills the singlehousing 12017. Accordingly, the elements of the equipment compartmentsand sample compartments are subjected to the introduced gas in theatmosphere within the housing 12017.

Unlike cooling air, which is conventionally drawn into the chamber tocool equipment, the introduced gas may be initially drawn into thechamber. Once the atmosphere of the housing 12017 is sufficientlystabilized, flow of the introduced gas may be terminated. Accordingly,sample incubation and measurement may then take place in a stableenvironment, in which both temperature and atmospheric concentration areprecisely controlled.

Implementation of housing 12017, dual-purpose base structure, fans, andother elements for controlling temperature of the UMS 12001 andtemperature and composition of the atmosphere within the housing 12017is not limited to the configuration illustrated in FIGS. 12 and 13.Rather, for example, the UMS 100 of FIG. 2 may be modified to furtherinclude such temperature and atmosphere control elements. Similarly, thetemperature and atmosphere control elements may be incorporated into thevarious embodiments of FIGS. 2 to 11 discussed throughout theapplication.

The housing 12017 illustrated in FIGS. 12 and 13 may be a clam shelldesign, consisting of two half enclosures that are mated together aroundthe main mechanical assembly. The main mechanical assembly by besuspended in the enclosed housing 12017 when both halves of the clamshell are mated together. To ensure that the completed housing 12017 isessentially gas tight, for example, a gasketing material could be usedon the interface between the two halves. The use of the gasketingmaterial is not limiting, as other forms of sealing may be employed.

The controlled environment of FIGS. 12 and 13 enables controlled ambientconditions, such as temperature and/or atmospheric concentration (e.g.,CO₂ and O₂ concentration) around the samples. When multiple measurementmodalities including imaging studies are required, all dedicatedinstruments should to be placed into environmental chamber, whichcreates problems with access and a need for manual intervention during along term experiment. However, the controlled environment of FIG. 12permits easy long term, multifaceted experiments to be conducted.

As discussed above, exemplary embodiments of the present inventionprovide a Universal Multi-detection System for determining fluorescence,chemiluminescence, and/or light absorbance of a sample in a microplatethat allows the user to reconfigure and optimize the measurement systemfor a particular modality. The Universal Multi-detection System allowsthe user to choose the best measurement method for his assay. The usercan select interference filters for their low detection limits andability to run FP, TRF, and HTRF measurements with state of the artresults, dual monochromators for their wavelength flexibility andspectral scanning, or a combination of both filters and monochromators.Both the excitation spectral device 500 and the emission spectral device700 may contain interchangeable filter systems and monochromators.

In addition to providing unmatched flexibility, the UniversalMulti-detection System also opens an avenue to run experiments that werenot previously possible. For example, in the fluorescence mode, whenonly a small amount of an unknown fluorofore is available and it is notpossible to increase the signal in the monochromator-based system byincreasing the sample concentration, the user can obtain roughexcitation and emission scans by using monochromators 530 and 720 in theexcitation spectral device 500 and the emission spectral device 700,respectively. Based on these initial excitation and emission scans, theuser can then select an appropriate filter 521 to excite the sample withlight that causes much stronger emission from the sample. The strongeremission spectrum can then be re-recorded. The user can also select afilter 711 to replace the emission monochromator 720 and re-record theexcitation spectrum. It is important to have high-quality measurementsof both the excitation spectrum and the emission spectrum. This processincreases the emission signal and the signal-to-noise ratio, resultingin improved excitation and emission spectra, as compared with spectraobtained with just a monochromator-based system. It also allows a userto work with an unknown sample, and optimize the measurement conditionsfor that sample.

This approach can also be used to achieve maximum sensitivity inend-point reads. The user can select particular excitation wavelengthsby using a monochromator 530 in the excitation spectral device 500, andtransmit the emission light through a filter 711 in the emissionspectral device 700. A similar benefit can be obtained by using a filter521 in the excitation spectral device 500 with a monochromator 720 inthe emission spectral device 700 during an end-point read. These methodsare particularly suited to enhancing performance of environmentallysensitive labels, where variations in conditions give rise toperturbations of excitation or emission spectra, such as ion sensitiveprobes, pH sensitive probes, spectral shifts in polar dyes withconjugation, and binding and membrane probes.

FIG. 17 is a functional block diagram that illustrates the control ofmodalities of a UMS, according to an exemplary embodiment.

As illustrated in FIG. 17, operation of the modalities of the UMS may becontrolled by a central control unit (e.g., processor, CPU,microprocessor, etc.). The processor may be connected to communicatewith and control elements of the sample environment, elements of sampleselection and positioning, elements of the monochromator module,elements of the filter module, and elements of the imager module.

Elements of the sample environment under control may provide temperaturecontrol and gas control, as discussed above.

Sample positioning may be controlled through the use of motors forpositioning samples in any of X and Y directions.

Elements of the monochromator module under control may includemonochromator excitation, monochromator emission, monochromator PMT, anda light source such as a flash lamp.

Elements of the filter module under control may include the filterselector, a filter PMT, and a light source such as a flash lamp.

Elements of the imager module under control may include an objectiveselector, an image capturing device such as a camera, a focus driveimager, and an LED selector and control.

FIG. 18 is a flowchart of control method of a UMS, according to anexemplary embodiment.

Control of the UMS may be coordinated through use of the processor, asdiscussed above with respect to FIG. 17. Input to the UMS (step S1805)may be accomplished through a local user interface of the UMS, such as atouch pad, or through communication with the UMS over a wired orwireless connection, such as over a network.

In the case of input to the UMS, input may be performed through the useof a user interface or graphical user interface displayed on a computeror other terminal that executes a control application.

The input may be user input, such as setting and parameters forexecuting control of the UMS.

In response to receiving input, control of the UMS may be effectuatedthrough the various elements of the UMS, discussed above regarding FIG.17. For example, in response to receiving user input, the UMS may becontrolled to execute a gas control procedure of the gas module (stepS1810), a sample positioning control procedure to control positioning ofsamples (step S1820), a monochromator and/or filter control procedure tocontrol operations of the monochromator and/or filter (step S1830), animager control procedure to control the imager (step S1840), and tooutput a result of the controlling of the elements of the UMS (stepS1850).

Although control is presented as illustrated in FIG. 18, elements may beindividually controlled in any sequence, and control of all elements isnot required. Accordingly, the multiple modalities of the UMS may becontrolled in a single assay. Additional aspects of the control of theUMS will be discussed below with respect to FIGS. 19 to 29.

The control method illustrated in FIG. 18 may be implemented throughexecution of a processing unit (e.g., CPU) controlling elements of theUMS by executing one or more control programs. The programs may bestored in a memory (i.e., RAM, ROM, flash, etc.), or othercomputer-readable medium (i.e., CD-ROM, disk, etc.). The program may beexecuted locally by the UMS, or by a control apparatus, such as acomputer that transmits commands to be executed by the UMS.

FIG. 19 illustrates a control apparatus for controlling a UMS, accordingto an exemplary embodiment.

As discussed above with respect to FIGS. 17 and 18, the UMS may becontrolled through communication with a control apparatus. The controlapparatus may be a laptop computer, as illustrated in FIG. 19.Communication between the UMS and the control apparatus may be conductedlocally through a USB cable, as illustrated in FIG. 19.

The control apparatus is not limited to the laptop computer, but may beany apparatus including a processor that executes control software forproviding a user interface (UI) for controlling operations of the UMS.The control software may be installed on the control apparatus, orexecuted by the control apparatus in communication with a server devicethat hosts the control software, for example over a network such as theInternet.

The control apparatus executing the control software may issue commandsto the UMS over the USB connection, though communication may beperformed using other wired techniques, such as Ethernet, or wirelesstechniques such as infrared or wireless communication. The communicationmay be direct, for example over the USB connection, or accomplishedthrough a network of intermediary devices, such as routers and switches.The network may be a local network, such as a local area network (LAN),or over a public network, such as the Internet.

FIG. 20 illustrates operations of an imaging subsystem of a UMS,according to an exemplary embodiment.

As discussed above, the UMS may include an imaging subsystem for imaginga sample.

The control software may include a UI for the imaging subsystem. The UIfor the imaging subsystem may receive user inputs for controlling theimaging subsystem to image a sample.

As illustrated in FIG. 20, the UI for the imaging subsystem may controlvarious aspects of the imaging subsystem including, but not limited to,parameters such as color, lamp power, and lens magnification.

The UI for the imaging subsystem may further select at least one well ata position of a microplate and a position of imaging a particularportion the well. As illustrated in FIG. 19, the imaging subsystem maycontrol the UMS to image individual samples one at a time, but may alsobe controlled to image an entire microplate.

Other options of the UI for the imaging subsystem may be used to controlfocus, signal, and digital zoom.

The settings of the imaging subsystem may be stored in memory, andretrieved for subsequent imaging of additional samples.

The UI for the imaging subsystem may be employed to view live samples inreal-time, review images of previous samples, and view reports of sampleimages.

As illustrated in FIG. 19, the imaging subsystem may control the UMS toimage individual samples one at a time, but may also be controlled toimage an entire microplate.

FIG. 21 illustrates mode selection of operations of a UMS, according toan exemplary embodiment.

As discussed above, the UMS may include an analysis subsystem foranalyzing a sample and an imaging subsystem for imaging the sample.

FIG. 21 illustrates the configuration in which the imaging by theimaging subsystem is selected. Naturally, other modes of the analysissubsystem, such as absorbance, luminescence, or the like, may beselected.

When selecting an analysis mode, the optics may also be selected by theuser, such as monochromator or filter. Alternatively, the optics may beautomatically selected based on the mode selection.

A read type may also be selected, such as endpoint/kinetic, spectralscanning, or area scanning Again, the read type may be selected by theuser or automatically selected based on the mode selection.

FIG. 22 illustrates parameter settings of an imaging subsystem of a UMS,according to an exemplary embodiment.

Upon selection of a detection mode, additional parameters may be set.

In the case of selecting the imaging mode illustrated in FIG. 21, theparameters for the selected imaging mode are shown in FIG. 22.

The selectable parameters may include color, lens, focus, gain, andadditional options, which are associated with the selected mode. Animaging speed and color for sample imaging may also be selected perwell, or for the entire microplate.

FIG. 23 illustrates parameter settings of an analysis subsystem of aUMS, according to an exemplary embodiment.

As discussed above, the UMS may include an analysis subsystem foranalyzing a sample and an imaging subsystem for imaging the sample.

FIG. 23 illustrates the configuration in which the analyzing by theanalysis subsystem is selected. While the detection mode selected inFIG. 23 is the fluorescence intensity analysis, other modes of theanalysis subsystem, such as absorbance, luminescence, or the like, maybe selected.

When selecting an analysis mode, the optics may also be selected by theuser, such as monochromator or filter. Alternatively, the optics may beautomatically selected based on the mode selection. In the case of thefluorescence intensity analysis, the optics for luminescence fiber andimaging may be automatically deselected (e.g., grayed out).

FIG. 24 illustrates parameter settings of an analysis subsystem of aUMS, according to an exemplary embodiment.

Upon selection of a detection mode, additional parameters may be set.

In the case of selecting the fluorescence analysis mode illustrated inFIG. 23, the parameters for the selected fluorescence mode are shown inFIG. 24.

The selectable parameters may include excitation, emission, opticspositioning, gain, and additional options, which are associated with theselected fluorescence analysis mode. A read speed, read height, and awavelength for sample analysis may also be selected per well, or for theentire microplate.

FIG. 25 illustrates settings of multiple subsystems of a UMS, accordingto an exemplary embodiment.

As discussed above, in addition to the imaging and analysis subsystems,the UMS may also include a temperature subsystem and a gas controlsubsystem.

FIG. 25 illustrates a procedure in which an assay temperature is set to37 degrees Celsius by control of the temperature control subsystem, acarbon dioxide level is set to 5% by control of the gas controlsubsystem, a reagent is automatically dispensed, then shaking happensfor 10 seconds, a kinetic measurement is initiated to automaticallyfollow fluorescence and image changes over a 10 hour time period every15 minutes, according to controls of the imaging and analysissubsystems.

Additional controls may be provided for independently controlling thevarious subsystems. For example, control operations of the varioussubsystem operations may be changed, paused, stopped, resumed, orscheduled based on a delay or timer.

FIG. 26 illustrates monitoring operations subsystems of a UMS, accordingto an exemplary embodiment.

As illustrated in FIG. 26, subsystems may be programmed by the user. InFIG. 26, fluorescence signal is monitored automatically, and when apre-set condition is met, imaging starts. Based on the pre-set conditionprogrammed by the user, only wells of interest matching the conditionmay be imaged.

The programming conditions may be stored to memory, and recalled frommemory for use with additional samples. Alternately, the programmingconditions may be modified as needed, and stored as additionalprogramming conditions.

Default programming conditions may be provided in a UMS, for selectionby a user according to frequently used programs. Alternately, newprogramming conditions may be adopted according to input of the user.

FIG. 27 illustrates results of operations of an analysis subsystem of aUMS, according to an exemplary embodiment.

As discussed above, programming conditions may be input by the user andthe programming conditions may be carried out by the UMS.

FIG. 27 illustrates results obtained from executing the programmingconditions illustrated in FIG. 26.

As illustrated in FIG. 27, wells have been read using standardfluorescence measurement, wells with initial result above 22,000 areautomatically imaged, and a thumbnail image is displayed for thesewells, in combination with the fluorescence result.

Alternately, as opposed to each well, only those wells satisfying thepre-set conditions may be displayed, thereby increasing viewingefficiency.

The use of such a programmed process allows for faster reading of sampleand limiting the size of the final data file since only the relevantwells are imaged.

FIG. 28 illustrates a detailed result of operations of an analysissubsystem of a UMS, according to an exemplary embodiment.

As discussed above, a programmed process may be executed by the UMS andresults may be cumulatively displayed.

As illustrated in FIG. 28, the results may be selectively displayed. Forexample, a “well zoom” may be displayed when clicking on one of theimage thumbnails.

In addition to the zoom image of the well, other statistics, graphs,charts, and raw data of the well may be presented.

In addition to display of results, data reduction tools may be provided,as shown in FIG. 29.

For example, “Image Analysis” tools may be provided for object counting,transfection efficiency, and total intensity. As illustrated in FIG. 29,“Cell Counting” based on image analysis may be an exemplary tool.

Exemplary embodiments of the present invention have been described forillustrative purposes, and those skilled in the art will appreciate thatvarious modifications, additions and substitutions are possible withoutdeparting from the scope and spirit of the invention as disclosed in theaccompanying claims. Therefore, the scope of the present inventionshould be defined by the appended claims and their legal equivalents.

1. A device for analyzing one or more samples, comprising: a support fora receptacle that holds a sample; an imaging subsystem that images thesample; and an analyzing subsystem that analyzes the sample.
 2. Thedevice of claim 1, wherein the analyzing subsystem comprises at leastone of a first measuring device that measures an absorbance of thesample, a second measuring device that measures a fluorescence of thesample, and a third measuring device that measures a luminescence of thesample.
 3. The device of claim 2, wherein the analyzing subsystemcomprises the first measuring device that measures the absorbance of thesample, the second measuring device that measures the fluorescence ofthe sample, and the third measuring device that measures theluminescence of the sample.
 4. The device of claim 2, wherein theanalyzing subsystem comprises at least two of the first measuring devicethat measures the absorbance of the sample, the second measuring devicethat measures the fluorescence of the sample, and the third measuringdevice that measures the luminescence of the sample.
 5. The device ofclaim 2, wherein the analyzing subsystem further comprises: at least oneof (i) a first transmission path that includes a filter through whichlight is transmitted to the sample and (ii) a second transmission paththat includes a monochromator through which light is transmitted to thesample.
 6. The device of claim 5, further comprising: a gas controlmodule that controls a composition of the atmosphere within a housing ofthe device.
 7. The device of claim 2, wherein the analyzing subsystemfurther comprises: (i) a first transmission path that includes a filterthrough which light is transmitted to the sample and (ii) a secondtransmission path that includes a monochromator through which light istransmitted to the sample.
 8. The device of claim 7, further comprising:a gas control module that controls a composition of the atmospherewithin a housing of the device.
 9. The device of claim 8, wherein thereceptacle support, the analyzing subsystem, the imaging subsystem, the(i) first transmission path and the (ii) second transmission path, andthe gas control module are integrated in the device.
 10. The device ofclaim 9, wherein the receptacle support, the analyzing subsystem, theimaging subsystem, the (i) first transmission path and the (ii) secondtransmission path, and the gas control module are contained in a housingof the device.
 11. The device of claim 1, wherein the receptaclecomprises a plurality of wells arranged in a matrix.
 12. The device ofclaim 1, wherein the receptacle support, the analyzing subsystem, andthe imaging subsystem are integrated in the device.
 13. The device ofclaim 12, further comprising: a housing that houses the receptaclesupport, the analyzing subsystem, and the imaging subsystem.
 14. Thedevice of claim 13, further comprising: a positioning subsystem thatrelatively positions the receptacle support and the analyzing subsystemto a first position within the housing at which the analyzing subsystemanalyzes the sample and that relatively positions the receptacle supportand the imaging subsystem to a second position within the housing atwhich the imaging subsystem images the sample.
 15. The device of claim14, wherein the positioning system relatively positions the receptaclesupport based on an index that identifies positions of a plurality ofwells of the receptacle.
 16. The device of claim 14, wherein thepositioning system comprises a carriage that moves the receptaclesupport.
 17. The device of claim 13, further comprising: an incubationchamber within the housing that incubates the sample.
 18. The device ofclaim 16, wherein the imaging subsystem comprises an objective that ismovably supported to be inserted into and removed from the incubationchamber.
 19. The device of claim 12, wherein the imaging subsystemcomprises an objective.
 20. The device of claim 19, wherein theobjective comprises a plurality of objectives mounted on a turret thatare selectable.
 21. The device of claim 18, wherein the imagingsubsystem images in at least one of a broad field mode and anepifluorescence mode.
 22. The device of claim 19, wherein the imagingsystem comprises an imaging light source and the analyzing subsystemcomprises at least one separate analyzing light source.
 23. The deviceof claim 22, wherein the analyzing subsystem measures at least one ofabsorbance of the sample and fluorescence of the sample illuminated withlight from the analyzing light source.
 24. The device of claim 12,wherein at least one of atmospheric temperature and atmosphericcomposition around the sample are controlled within a housing of thedevice.
 25. The device of claim 17, wherein the atmospheric temperaturewithin the incubation chamber is controlled and the gas compositionwithin the housing is controlled.
 26. The device of claim 13, whereinone of (i) the imaging subsystem and the analyzing subsystem and (ii)the receptacle support share a commonly controlled atmosphericcomposition within the housing.
 27. The device of claim 26, wherein boththe imaging subsystem and the analyzing subsystem share the commonatmospheric conditions.
 28. The device of claim 24, further comprising:a gas control subsystem that controls the atmospheric composition withinthe housing.
 29. The device of claim 28, wherein the gas controlsubsystem comprises: at least one gas sensor; and at least one gascontrol valve.
 30. The device of claim 28, further comprising: atemperature control subsystem that controls the atmospheric temperatureat least within an incubation chamber within the housing.
 31. The deviceof claim 30, wherein the receptacle support, the analyzing subsystem,the imaging subsystem, the gas control subsystem, and the temperaturecontrol subsystem are integrated in the device.
 32. The device of claim31, further comprising: a processor that controls operations of theanalyzing subsystem, the imaging subsystem, the gas control subsystem,and the temperature control subsystem.
 33. The device of claim 32,further comprising: a common interface operated by a user to control theoperations of the analyzing subsystem, the imaging subsystem, the gascontrol subsystem, and the temperature control subsystem.
 34. The deviceof claim 11, further comprising: a temperature control subsystem thatcontrols a temperature of an atmosphere at least within an incubationchamber of the device.
 35. The device of claim 34, wherein thetemperature within the housing is maintained at about ambienttemperature by: a plate that conducts heat from an atmosphere within thehousing; a fan outside the housing that cools the plate through forcedconvection.
 36. The device of claim 1, further comprising: a processorthat controls operations of the analyzing subsystem and the imagingsubsystem.
 37. The device of claim 36, further comprising: a commoninterface operated by a user to control the operations of the analyzingsubsystem and the imaging subsystem.
 38. A sample examination methodcomprising: selecting at least one subsystem from among a plurality ofsubsystems of a sample analysis device that examines one or moresamples, the plurality of subsystems comprising an imaging subsystemthat images the one or more samples and an analyzing subsystem thatanalyzes the one or more samples; and controlling the selected at leastone subsystem to perform an examination on the one or more samples, theexamination comprising an imaging operation of the imaging subsystemthat images the one or more samples and an analyzing operation of theanalyzing subsystem that analyzes the one or more samples.
 39. Themethod of claim 38, further comprising: displaying a result of thecontrolling the selected subsystem to perform the examination on adisplay.
 40. The method of claim 38, wherein the at least one subsystemcomprises the imaging subsystem and the analyzing subsystem, wherein theselecting comprises selecting the imaging subsystem and the analyzingsubsystem, and wherein the controlling comprises controlling the imagingsubsystem to perform the imaging operation on the one or more samplesand controlling the analyzing subsystem to perform the analyzingoperation on the one or more samples.
 41. The method of claim 38,wherein the selecting comprises: displaying the plurality of subsystemson a user interface; and receiving an input of a user that selects theselected at least one subsystem from the displayed plurality ofsubsystems.
 42. The method of claim 38, wherein the plurality ofsubsystems further comprises a gas control subsystem that controls anatmospheric composition of an atmosphere within a housing of the sampleanalysis device.
 43. The method of claim 38, wherein the plurality ofsubsystems further comprises a temperature control subsystem thatcontrols an atmospheric temperature of an atmosphere within a housingthe sample analysis device.
 44. The method of claim 43, wherein thetemperature control subsystem controls the atmospheric temperature ofthe atmosphere within an incubation chamber within the housing.
 45. Themethod of claim 38, wherein the analyzing subsystem comprises at leastone of a first measuring device that measures an absorbance of thesample, a second measuring device that measures a fluorescence of thesample, and a third measuring device that measures a luminescence of thesample.
 46. The method of claim 38, wherein the analyzing subsystemcomprises at least one of (i) a first transmission path that includes afilter through which light is transmitted to the sample and (ii) asecond transmission path that includes a monochromator through whichlight is transmitted to the sample.
 47. The method of claim 38, furthercomprising: relatively positioning a receptacle support of the at leastone or more samples and the selected subsystem to a position at whichthe selected subsystem performs the examination on the one or moresamples.
 48. The method of claim 47, wherein the positioning comprises:identifying positions of the at least one or more samples based on anindex that identifies positions of the at least one or more samples in areceptacle supported by the receptacle support; and relativelypositioning the receptacle support based on the identified positions.49. The method of claim 38, wherein the imaging operation comprises:selecting an imaging objective from among a plurality of imagingobjectives; and imaging the at least one or more samples using theselected imaging objective.
 50. A non-transitory computer-readablemedium having embodied thereon a program which when executed by acomputer causes the computer to execute a sample examination method, themethod comprising: selecting at least one subsystem from among aplurality of subsystems of a sample analysis device that examines one ormore samples, the plurality of subsystems comprising an imagingsubsystem that images the one or more samples and an analyzing subsystemthat analyzes the one or more samples; and controlling the selected atleast one subsystem to perform an examination on the one or moresamples, the examination comprising an imaging operation of the imagingsubsystem that images the one or more samples and an analyzing operationof the analyzing subsystem that analyzes the one or more samples.